Preparación de las estructuras monolíticas de carbón

While we were able to identify significant numbers of p16INK4a_{-expressing cells}

from several tissues with aging, the low frequency of tdTom+ cells (<10%) and difficulty of isolating these fractions prevented us from further functional and molecular

characterization. Therefore, we turned to a recently described inflammatory model to induce high-level p16INK4a expression in activated macrophages in vivo (42). Toward that end, we implanted quiescent neonatal dermal fibroblast (NDF)-containing alginate beads into p16LUC/+ or p16tdTom/+ mice via intraperitoneal injection. Prior work has shown that these quiescent NDFs quickly acquire SA-β-gal staining and release soluble factors

including IL-6 and IL-8, in turn leading to a large influx of inflammatory cells (42). As reported, NDF beads induced a strong luminescent signal in the abdomen of p16LUC/+ mice by 3 weeks post-injection (Fig. 2.5A). Flow cytometric analysis of cells in the peritoneal lavage of p16tdTom/+ _{mice 3 weeks after implanting NDF beads showed a}

strong induction of tdTom expression in macrophage (Mac-1+F4/80+) populations (Fig. 2.5B), but not other lavage cell types (e.g. T cells, B220+ cells and eosinophils, Fig. S2.6). To characterize peritoneal macrophages with high-level p16INK4a promoter

activation, we isolated Mac-1+F4/80+ cells by FACS based on tdTom expression. Using this approach, we observed a much greater enrichment of p16INK4a _{mRNA expression in}

tdTom+ vs. tdTom- macrophages (40-fold, Fig. 2.5C) compared to that seen in MEFs (5-fold, Fig. 2.2B). This likely reflects much greater homogeneity among the sorted macrophages compared to mixed MEF cultures. As was the case for MEFs, tdTom+ macrophages exhibited a marked reduction in EdU incorporation (Fig. 2.5D) and increased SA-β-gal activity (Fig. 2.5E-F). It is worth noting that SA-β-gal activity has

been considered an unreliable marker of senescence in vivo, especially in this cell type (43, 44). These results show that a substantial fraction of macrophages induced in response to NDF-loaded beads exhibit features of senescence: activation of the p16INK4a promoter, reduced proliferation and expression of SA-β-gal activity.

Prior studies suggest that p16INK4a also influences cell intrinsic properties of macrophages, such as M1/M2 polarization (45, 46). In order to further investigate the effect of p16INK4a on macrophage function, we examined the immunophenotype and cell-specific functions of p16INK4a-activated macrophages in more detail. We did not observe a difference in the immunophenotype of tdTom+ versus tdTom- lavage cells

with regard to macrophage polarity (e.g. CD80, CD206 and MHCII). Moreover, we did not find a modulation of p16INK4a promoter activity by M1/M2 polarizing agents including LPS and interleukin 4 (IL-4) in either tdTom+ or tdTom- macrophages (Fig. S2.7). However, in vitro phagocytosis assays showed that tdTom+ macrophages exhibited greater phagocytic activity than tdTom- cells (Fig. 2.6A-B). This demonstration of altered or even increased cellular function is reminiscent of findings in other cell types in the setting of high-level p16INK4a expression (e.g. increased insulin secretion from p16INK4a- expressing pancreatic beta cells (47) and increased cell killing in senescent T cells (46, 48)).

In order to study the underlying mechanisms and genes responsible for the response of p16INK4a_{-activated macrophages to NDF-beads, we performed RNA-seq of}

tdTom+ versus tdTom- peritoneal macrophages. We identified 456 transcripts being upregulated and 118 transcripts downregulated in tdTom+ macrophages (P<0.01). Through gene set enrichment analysis (GSEA), we identified several gene signatures related to the cell cycle, senescence and macrophage functions (Figure 2.6C-E). Specifically, consistent with the decreased proliferation of these cells (Fig. 2.5D), tdTom+ macrophages exhibited a profound decline in the expression of transcripts associated with cell cycle traversal and ribosomal proteins. Even though expression of a few “cell cycle”-classified genes was increased in tdTom+ cells, these were largely inhibitors of the cell cycle such as p16INK4a/Arf (Cdkn2a) and p15INK4b (Cdkn2b, Fig. 2.6E). Macrophages with high-level activation of the p16INK4a _{promoter also exhibited}

increased expression of lysosomal mRNAs, consistent with the observed increase in β-

did not observe differential expression of genes associated with M1/M2 macrophage polarization (e.g. Nos2, Arg1 and Ym1/2). On the other hand, we found increased expression of genes involved in phagocytosis in tdTom+ macrophages (Fig. 2.6D), consistent with the high phagocytic activity of p16INK4a_{-activated macrophages (Fig.}

2.6A-B). Additionally, we found clear upregulation of several components and regulators of the extracellular matrix (ECM) or the “matrisome” including collagens, matrix

metalloproteinases, thrombospondins, and fibulins (Fig. 2.6C and E), and these

changes were highly consistent with prior studies of the ECM in senescent cells (35, 49). Finally, using the list of SASP transcripts developed for the MEF RNA-seq studies (Fig S2.4 and Table S2.1), we showed a strong enrichment by GSEA for SASP transcripts in tdTom+ cells (e.g. IL7, Mmp12, Timp2, Cxcl12/13, Hgf) while only one SASP transcript, Mif was expressed at lower levels (Table S2.1).

Contrary to less clear results generated from heterogeneous MEFs, these studies were carried out in a well-defined in vivo hematopoietic subtype and

demonstrate that p16INK4a-activated macrophages exhibit multiple features of senescent cells by gene expression analysis including decreased replication, increased lysosomal activity, altered ECM production and expression of many classic SASP factors.